人巨细胞病毒pp65基因在毕赤酵母中的表达

Expression of Human Cytomegalovirus pp65 Gene in Pichia pastoris

目的构建表达人巨细胞病毒(HCMV)pp65336-507基因的毕赤酵母工程菌。方法扩增HCMVpp65336-507基因,构建重组表达质粒pPIC9/pp65336-507,转化毕赤酵母菌GS115。PCR法筛选阳性克隆,SDS-PAGE及Westernblot鉴定表达产物。结果已构建和筛选到阳性表达克隆株,表达的pp65336-507蛋白的相对分子质量约为26000,能与特异的pp65抗体结合。结论已成功构建了分泌表达pp65336-507蛋白的毕赤酵母工程菌,表达的目的蛋白具有良好的反应原性。

Objective To construct a recombinant Pichia pastoris for expression of human cytomegalovirus （HCMV） pp65336-507 gene. Methods Amplify HCMV pp65336-507 gene from the genome of HCMV by PCR using the designed primers to construct recombinant plasmid pPIC9/pp65336-507. Transform P. pastoris GS115 with the constructed recombinant plasmid, screen positive clones by PCR, and identify the expressed product by SDS-PAGE and Western blot. Results Positive recombinants were successfully screened. The expressed pp65336-507 protein, with a relative molecular mass of about 26 000, reacted with the specific antibody against pp65, Conclusion The recombinant Pichia pastoris for secretory expression of pp65336-507 protein was successfully constructed, and the expressed product showed good reactogenicity.